There are an esfimated 10,000 human single-gene disorders, which impose a significant burden on human health woridwide. The 5-year goals of this NDC are to develop a clinically applicable gene correction technology to treat single-gene disorders, and to demonstrate the efficacy of this approach in treating sickle cell disease (SCD) using a mouse model. SCD is caused by a single (A-T) mutafion in the beta-globin gene;it is a painful and life shortening disease and afflicts primarily persons of African origin. To develop the gene correcfion approach for treafing SCD, we will engineer and opfimize zinc finger nuclease (ZFN) proteins that bind specifically to the beta-globin gene, deliver them as well as wild-type donor templates into the nuclei of hematopoiefic stem cells (HSCs) to induce a DNA double strand break (DSB) or a nick at a preselected site near the beta-globin locus, shepherding the broken DNA ends into the homologous recombination (HR) pathway for gene correction. The autologous gene-corrected HSCs will be re-engrafted in a mouse model of SCO to produce healthy red blood cells and replace sickle cells. HSCs are the normal precursors of all blood cells, including the oxygen-carrying erythrocytes rendered dysfuncfional in sickle cell patients. These cells are relatively rare in the body, but possess potent regenerative potential in that transplantation of even a single HSC is sufficient to rebuild the entire blood system of an organism. Thus, by isolafing HSCs that carry the sickle mutafion, correcfing this mutafion ex vivo, and then transplanfing the gene-corrected HSCs back into affected recipients, we would be able to provide enduring replacement of the blood-producing cells of SCD pafients with unaffected precursors, thereby supplying healthy red blood cells (RBCs) and effectively curing the disease. Our proposed research represents a significant paradigm shift in current gene correction/gene therapy approaches in that no virus or foreign DNA is used. There are many pracfical and technological challenges in achieving our goals, including increasing the spontaneous rate of gene correction by many orders of magnitude, achieving high specificity and temporal control of ZFN activity, highly efficient delivery of ZFN and template delivery, and avoiding or rejecfing unwanted mutafions and gene rearrangements. We propose to overcome these challenges using nanotechnology and nanomedicine approaches, which will allow us to observe, control, and systemafically optimize each step in the gene correction process. The team will also explore scale-up and IND/IDE issues such as safety, high throughput delivery and quality control, with the goal of being ready to begin clinical trials at the end of the 5-year project period.